This research was supported by NIH Grant AG043080 and the National Natural Science Foundation of China (NSFC), No. 11434001. We thank Lucas Waldburger for proofreading the manuscript.

1.硅片模具制造注:光掩膜的设计与AutoCAD软件和商业公司制造的。这种设计包含不同的图案（三层<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55412/single_cell_trap.dwg" target="_blank">补充文件1</a> ）。第一，第二，和第三层的高度分别为大约4微米，10微米和50微米。硅晶片模具从使用软光刻9,10光掩模创建。 <ol><li>在200℃下烘烤的硅晶片10分钟，以蒸发水蒸汽。旋涂负性光刻胶SU-8到硅晶片上。 注意:旋涂SU-8 3005在5,000rpm下30秒，以产生第一层，SU-8 2010以3,000rpm进行30秒，以产生第二层，和SU-8 3025以2000rpm进行30秒，以产生第三层。 </li><li>软烤吨处理前的涂布晶片他的图案转移。对准，并且使用掩模对准器在暴露"直接接触"模式的晶片。 注:软烘烤的晶片在95℃下2分钟，所述第一层，在95℃下3分钟为第二层，和在95℃下15分钟的第三层。在100mJ / cm 2的第一层，130毫焦耳/厘米2的第二层，和为200mJ / cm 2的第三层使用的曝光剂量。 </li><li>曝光后，后烘烤的晶圆，并使用SU-8开发者开发。干燥使用N 2枪和硬烘烤在200℃下将晶片30分钟的晶片。 注:将硅晶片模具是用透明胶带固定到15厘米直径的塑料培养皿中，与图案的一面朝上。一般，我们把在相同的模具，它允许多个微流体芯片来同时制造多个相同的芯片结构。每个模具可以重新使用多次以制造微流控芯片。 </li></ol> 2.微流控芯片制造<ol><li>放在天平上的清洁称重船和去皮。倾50克PDMS碱入称量皿。 </li><li> 5克PDMS固化剂添加到所述称量皿（w / w至所述PDMS基的1:10的比例）。 注意:此卷是基于一个直径为15cm，培养皿与模具。如果使用不同大小的培养皿中调整试剂的量。 </li><li>搅拌PDMS基并用一次性移液管的固化剂。从称量船的边缘开始慢慢地向内移动。充分搅拌几分钟直至小气泡形成在整个混合物中;彻底混合对于PDMS聚合是必不可少的。 </li><li>把混合物慢慢倒入培养皿;该混合物必须完全覆盖硅晶片模具。 </li><li>放置在真空中的陪替氏培养皿10分钟，以除去从PDMS混合物中的所有气泡。如果气泡保留在混合物的表面上，用移液管吹出来。 </li><li>孵育硅晶片与模具在75℃的烘箱中用于PDMS约2小时。 </li><li>轻轻剥离聚合PDMS断硅晶片模具的层，避免对晶片模具的结构的任何损坏。可替换地，直接地从硅晶片与模具周围使用单边缘工业刀片图案的最小5毫米余量切割PDMS;轻轻剥离PDMS层从晶片模具。 </li><li>放置PDMS层与图案面朝上的替补​​。仔细地切出具有单边缘工业刀片的单独的芯片，保持从每个单芯片，以避免损害结构的边缘足够的裕度。 </li><li>朝上的图案的一面，使用冲头笔（0.75毫米ID），以打孔通过入口和出口圆直倒在通道的每一侧。 注意:这些孔创建介质流动的通路。因此，关键是要经过圈，并通过PDMS层冲一路。确保从孔去除PDMS列。 </li><li>通过再次插入冲笔针头插入孔检查每一个冲孔。确保针头可以从另一面出来，这表明没有堵塞。将胶带粘图案表面，然后轻轻剥离胶带清洁灰尘颗粒。重复此步骤至少三次。留在PDMS一块干净透明胶带，以保持消毒。 </li><li>重复对PDMS的相对侧此过程并离开最后一块胶带上为好。 </li><li>制备一个24毫米×30毫米玻璃盖的厚度为0.13-0.17毫米。喷洒70％的乙醇在玻璃上并干燥与除尘器消毒的表面上;另外，该玻璃可以与高压灭菌水洗涤，并用除尘器干燥。 </li><li>转移盖玻璃和PDMS的塑料板。从PDMS取下胶带并放置图案的一面朝上。避免在传送过程中与图案表面的任何接触。 </li><li&gt;放置在等离子体机的塑料板。应用氧等离子体处理的PDMS和玻璃盖片以使表面亲水性的具有下列操作参数:曝光，75秒;气体稳定，20毫升/分钟;压力，200;和电源，100 W. </li><li>小心地将PDMS到盖玻璃，连接两个亲水表面（即在等离子体处理期间朝上的表面）。确保有是PDMS和玻璃盖之间没有气泡。 </li><li>孵育在烘箱中在PDMS芯片在75℃下至少2小时。 注意:微流体缺陷可能会导致液体渗漏到在实验过程中的显微镜。因此，通过从与镊子的边缘略微抬起PDMS检查PDMS和盖玻璃之间的结合是重要的。轻轻提起PDMS不应它从玻璃盖分开，指示成功的键。 </li></ol> 3.实验准备<ol><l我&gt;管的准备。 <ol><li>淹没在70％乙醇溶液分别PDMS芯片制备前1天的输入和输出管。 </li><li>填充有5毫升的注射器用高压灭菌水清洗管。通过插入注射器上的管，并用水冲洗管洗每个管中。 </li><li>重复该洗涤步骤至少3次以清洁乙醇残留物。 </li></ol></li><li>检查用10X物镜在光学显微镜下的PDMS通道，以确保该结构是一致的和完整的。稳定PDMS芯片上使用透明胶带的显微镜平台。 注:在步骤2中的时间使多个芯片，以确保有备份芯片，特别是如果使设备首次。 </li><li>填充有5毫升的注射器用高压灭菌水。固定注射器的注射泵，并插入相应的输入和输出管。设置洗涤速度至750μL/ h至冲洗芯片约10分钟。在分支通道中的气泡应该在此期间被洗掉;如果有剩余的气泡，手动调整速度以消除气泡。 </li><li>酵母样品制备。 <ol><li>传输1制备的酵母样品（OD 600 0.6-0.8）中的溶液到两个1.5毫升微量离心管中离心5分钟，在3000×g下。 </li><li>除去大部分从每管中的上清液并结合其余以形成0.5毫升的样品。 </li></ol></li><li>暂停注射泵和从PDMS芯片删除输入管。 </li><li>手动反向注射泵吸小气泡（1至2cm长）到每个输入管。移动对酵母​​样品中吸;气泡将显示样品边界，并指出有多少样本已经绘就。 注意:避免将试料吸入到注射器中。 </li><li>插入输入管放回PDMS芯片，并重新启动泵以750μL/ h的速度加载细胞。 </li><li>离开注射器泵上10分钟的n和检查显微镜下细胞加载进展;一个成功加载将捕获细胞下暂停支柱的60％以上。 </li><li>切换到营养溶液和调整速度以用于细胞培养400微升/小时。 </li><li>在显微镜观察选择位置。 注:对于寿命测量，图像通过光学显微镜拍摄一次，每15分钟用40×物镜。对于荧光报告分析，图像通过用60X油浸物镜的荧光显微镜拍摄每15分钟一次。 </li></ol>

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The authors declare that they have no competing financial interests.

PDMS的设备需要新鲜的。否则，引起的插入管插入装置的空气气泡将难以去除。步骤3.4是重要通过浓缩细胞提高电池装载效率。为了增加实验的吞吐量，连接到同一个PDMS芯片上4至6个模块来独立地操作泵通常用于执行4至6个不同的实验（不同菌株或介质的组合物）同时进行。 相比于常规的酵母复制型老化试验（使用显微解剖），这里提出的微流体方法不太费力和耗时的。此外?...

芽殖酵母是衰老研究了强大的模式生物。然而，在酵母的常规寿命测定依赖于显微切割，这不仅是劳动密集，而且低吞吐量1,2。此外，他们的年龄在传统的显微方法不提供在单一母细胞的各种细胞和分子特征的详细视图。微流体装置的发展已使自动程序以测量酵母寿命以及跟随的分子标记和各种细胞表型在整个母细胞3，4，5，6，7，8的寿命。后的酵母细胞被加载到微流控装置，它们可以在显微镜下使用自动时间圈跟踪È成像。随着成像处理工具的帮助下，各种细胞和分子的表型可以被提取3，6，8，包括寿命，大小，荧光报道，细胞形态，细胞周期动力学， 等 ，其中许多是难以或不可能获得使用传统的显微解剖法。因为他们的成功发展了几年前3，4，6，7的微流体装置已经获得了在酵母衰老研究突出。一些研究小组已经随后发表在早期的设计5的变化，许多酵母实验室已经采用微流体装置作为研究对象。 在细胞培养进行指数增长，可用于观测岁母细胞的数量是miniscu乐。因此，微流体装置的寿命测量一般设计原则是保留母细胞，并删除子细胞。一个这样的设计使得使用酵母进行不对称细胞分裂的事实。在该装置将捕获更大母细胞的结构和更小的允许子细胞被冲走。这篇文章中描述的微流体芯片采用了软聚二甲基硅氧烷（PDMS）垫（垂直悬挂列）以捕获母细胞（ 图1）。类似的设计的设备先前已被3,4，6,7的报道。该协议使用一个简单的程序来制造微流体装置和被用于延时成像实验优化的直接的细胞装载方法。一个在微流体装置的关键参数是用于捕获母细胞的PDMS焊盘的宽度。我们ðevice使用更宽垫，可以让更多的母细胞各垫下，包括可以在整个他们的寿命进行跟踪新鲜母细胞的显著部分。除了寿命测量，当细胞需要跟踪许多代或当在整个寿命的观察是必要这个协议是用于单细胞延时成像实验是有用的。

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			<td>3&#39;&#39; &#60;111&#62; silicon wafer</td>
			<td>Addison Engineering</td>
			<td></td>
			<td></td>
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			<td>SU-8 2000 and 3000 Series</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
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			<td>SYLGARD&#174; 184 SILICONE ELASTOMER KIT</td>
			<td>ellsworth</td>
			<td>2065622</td>
			<td>Include Sylgard&#174; silicone elastomer base and curing agent</td>
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			<td>Petri dishes</td>
			<td>VWR</td>
			<td>391-1502</td>
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			<td>Harris Uni-core&#8482; punch(I.D. 0.75 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>29002513</td>
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			<td>24 mm x 40 mm SLIP-RITE&#174; cover glass</td>
			<td>Thermo Fisher Scientific</td>
			<td>102440</td>
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			<td>3M&#160; Scotch Tape</td>
			<td>ULINE</td>
			<td>S-10223</td>
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			<td>VWR&#174; Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
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			<td>PURE ETHANOL, KOPTEC</td>
			<td>VWR</td>
			<td>64-17-5</td>
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			<td>WHOOSH-DUSTER&#8482;</td>
			<td>VWR</td>
			<td>16650-027</td>
			<td></td>
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			<td>5mL BD Syringe (Luer-Lock&#8482;Tip)</td>
			<td>Becton, Dickinson and Company.</td>
			<td>309646</td>
			<td></td>
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			<td>PTFE Standard Wall Tubing (100ft, AWG Size:22, Nominal ID: 0.028)</td>
			<td>COMPONENT SUPPLY COMPANY</td>
			<td>SWTT-22</td>
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			<td>Needle Assortment</td>
			<td>COMPONENT SUPPLY COMPANY</td>
			<td>NEKIT-1</td>
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			<td>Desiccator</td>
			<td>HACH</td>
			<td>2238300</td>
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			<td>Lab Oven</td>
			<td>FISHER SCIENTIFIC</td>
			<td>13246516GAQ</td>
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			<td>Nikon TE2000 microscope with 40x and 60x objective</td>
			<td>Nikon</td>
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			<td>Zeiss Axio Observer Z1 with 40x and 60x objective</td>
			<td>Zeiss</td>
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			<td>Syringe Pump</td>
			<td>Longerpump</td>
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using microfluidic devices to measure lifespan and cellular phenotypes in single budding yeast cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

实验后，将细胞和许多细胞和分子表型的寿命可以从所记录的时间推移的图像被提取。由于有许多可从每个细胞中提取不同的特征，分析的第一步是将注释细胞和事件，包括位置和小区的边界和正被跟踪的各种事件的定时，例如作为萌芽事件。这些注释将使它更容易返回到相同的一组细胞并分析不同的特点，在未来。使用图像分析软件，如ImageJ的11和相?...

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Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

本文介绍用于生产微流体芯片和微流体实验的设置来测量的寿命和单个酵母细胞的细胞表型进行了优化的协议。

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

using-microfluidic-devices-to-measure-lifespan-cellular-phenotypes

Research

JoVE Journal

Biology

7.6K 次观看.  Peking University. 本文介绍用于生产微流体芯片和微流体实验的设置来测量的寿命和单个酵母细胞的细胞表型进行了优化的协议。 

视频: Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

科研

生物学

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

在裂殖酵母中Cytokinetic活动的时空分析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

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